Alanine screening of the intracellular loops of the human bradykinin B₂ receptor – effects on receptor maintenance, G protein activation and internalization

Abstract

The human bradykinin B 2 receptor (B 2 R) mediates the effects of the nonapeptide bradykinin (BK) and of kallidin (lysyl-BK). B 2 R has been reported to play a role in a number of physiological and pathophysiological situations. Its activation causes vasodilation and hypotension, increased vascular permeability and edema, or generation of pain via C fibers [1]. B 2 R, which is expressed constitutively in many tissues and cultured cells, is a prototypical member of family A (rhodopsin ⁄ b-adrenergic-like receptors) of the membrane-bound The bradykinin B 2 receptor is coupled to G protein G q ⁄ 11 and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY fi AAA, retained the ability to specifically bind [ 3 H]bradykinin. The binding affinities at 4 or 37°C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [ 3 H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in G q ⁄ 11 activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability.Abbreviations B 2 Rwt, bradykinin B 2 receptor wild-type; BK, bradykinin; CMV, cytomegalovirus; EC 50, half-maximal effective concentration; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; HEK 293, human embryonic kidney cells; ICL-1, ICL-2, ICL-3, first, second and third intracellular loops; IP, inositol phosphate; PAO, phenylarsine oxide.

Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

“…Cell Culture and Transfection-For stable expression of the constructs in HEK 293 cells we used the Flp-In system from Invitrogen as described previously (24). For transient transfection cells were seeded into 24 wells (80% confluency) and transfected using 0.2 g of DNA and 0.4 l of EcoTransfect.…”

“…Determination of Inositol Phosphate (IP) Release-Phosphatidylinositol hydrolysis was measured as previously described (24). In brief, confluent cell monolayers (24 wells) were incubated overnight in 0.5 ml of complete medium containing 1 Ci [ 3 H]inositol/ml.…”

“…After 30 min IP accumulation was stopped by exchanging the medium for 1.5 ml of ice-cold 20 mM formic acid solution. After 1 h on ice and application of the supernatant to AG 1-X8 columns, total [ 3 H]IP was determined as described before (24).…”

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B1 and B2 receptors

“…Expressing the B 1 R under the control of the strong cytomegalovirus (CMV) promoter and the B 2 R under the control of the weaker promoter P min led to comparable protein levels of the B 1 R and the B 2 R (1200-2000 fmol/mg protein) in our HEK 293 cells, as determined by radioligand binding assays (Faussner et al, 2009) and therefore similar signaling activity. However, to exclude that our observations resulted solely from this heterologous B 1 and B 2 receptor overexpression, we reproduced our findings in the human embryonic lung fibroblast cell line IMR 90, which endogenously expresses the B 2 R (300-800 fmol/mg protein).…”

“…Stimulation was terminated by exchange of ligand solution for 1.5/0.75 ml of ice-cold 20 mM formic acid solution. After 60 min, the amount of total IP in the formic acid extract was determined by column chromatography using AG 1-X8 columns (Bio-Rad, Munich, Germany) as described earlier (Faussner et al, 2009). …”